The studies planned for the coming year are directed toward the ultimate goal of determining the biological significance of nonbanding, homogeneously staining regions (HSRs) and the large numbers of double minute chromosomes (DMs) that characterize cells of 14 out of 16 different human neuroblastoma cell lines maintained in our laboratory. Specifically, we plan to continue two-dimensional gel electrophoretic analyses of proteins of human neuroblastoma cells and to make comparisons with several different tumor cell types. Although we have not yet identified candidate products of putatively amplified genes, results so far indicate considerable similarity in gel patterns between several different near-diploid neuroblastoma cell populations. Clones of HSRcontaining and HSR-lacking human-mouse neuroblastoma cell hybrids also will be studied by this approach in attempts to identify an HSR-associated gene product. We also plan to initiate experiments in which we will attempt to determine whether human neuroblastoma cells, in contrast to certain other types of human tumor cells, synthesize a neuroblastoma-specific factor to which neuroblastoma cells respond. The ability of target neuroblastoma cells to grow in defined media with added media conditioned by neuroblastoma and nonneuroblastoma cells will be tested initially. In addition, we plan to continue preliminary in situ hybridization experiments utilizing Cot-purified, amplified genomic DNA sequences from several human neuroblastoma cell lines as probes in order to determine whether all of the HSRs of a variety of different human neuroblastoma lines contain essentially the same set of amplified sequences. We will also determine whether putative abnormally banding regions (ABRs) are indicative of low level sequence amplification. We will also be cloning several Eco R1 restriction fragments in order that they may be used to carry out further Southern blot analysis of various human neuroblastoma cell lines, to do Northern blot analysis of the mRNA populations of these cell lines to determine whether any of them share sequence homology with poly A+ RNA and to screen genomic libraries in order to obtain overlapping DNA sequences, eventually enabling us to describe the structural nature of the amplification unit in these cells.